Florey Department of Neuroscience and Mental Health - Theses
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ItemCharacterization of white matter asymmetries in the healthy human brain using Diffusion MRI fixel-based analysis( 2020)Magnetic resonance imaging (MRI) has revolutionized the way to investigate brain structural connectivity non-invasively. Diffusion MRI can be used to obtain local estimates of the white matter fibre orientations in the brain, which in turn can be used to study changes in the local fibre specific properties and/or in conjunction with fiber-tracking algorithm to reconstruct a representation of the white matter pathways in the brain. In recent years, the Diffusion Tensor model has played an important role in modelling the diffusion of water within white matter bundles. Diffusion tensor derived metrics such as fractional anisotropy (FA) have been used extensively for investigating white matter using approaches such as voxel-based analysis. One of the limitations of the diffusion tensor model is that it is not capable of appropriately modelling regions that have complex fibre architecture (such as crossing fibres). This makes tensor-derived measures unreliable measures to assess the white matter. Recent contributions toward the study of brain asymmetry have suggested asymmetry of brain anatomy and function are observed in the temporal, frontal, and parietal lobes. Several studies have used diffusion tensor model to study asymmetry in various regions of the human brain white matter. However, given the limitations of the tensor model, the nature of any underlying asymmetries remains uncertain. This research aims to provide to provide a more robust characterization of structural white matter asymmetries than those previously derived using the tensor model, by using quantitative measures derived from the spherical deconvolution model, and a whole-brain data-driven statistical inference framework such as Fixel-Based Analysis, that is both sensitive and specific to crossing fibres; we furthermore apply this approach to a state-of-the-art publicly available diffusion MRI dataset.
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ItemThe effect of genetic susceptibility and immunosuppressant treatment on mononuclear cell phenotype in multiple sclerosis( 2019)Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The immune system plays a significant role in the pathophysiology of the disease and research into the mechanisms that may be potentially contributing to the disease is essential to help advance knowledge in the field and ultimately seek better outcomes for MS patients. A key set of cells that are believed to play a role in MS immunopathology are mononuclear cells which consist of B-cells, T-cells, monocytes and their derivatives. The aims of this thesis was to assess the effects of 1) genetic susceptibility and 2) immunosuppressant treatment, on mononuclear cell phenotype in MS. The first aim involved interrogating a key co-stimulatory molecule, cluster of differentiation 40 (CD40), expressed on mononuclear cells which is known to play a fundamental role in the regulation of the humoral immune response. CD40 is a risk gene for the development of MS and I sought to assess the effects of the single nucleotide polymorphism (SNP) rs1883832 on the phenotype of CD40-expressing mononuclear cells. Utilising a range of bimolecular methods and samples taken from MS individuals and healthy matched-controls (HC), I showed that SNP rs1883832, particularly the ‘risk’ genotype (homozygous TT) was correlated with reduced soluble CD40 (sCD40) levels in HC, but not MS patients. I did not observe any phenotype-dependent differences in sCD40 levels between MS (in quiescent state) and during active demyelination compared to HC. Upon assessment of key cytokines produced by B-cell and monocytes, I observed elevated gene expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in the MS population linked to the CD40 risk TT-genotype. In the second component, I showed that the immunosuppressant drug Cladribine, is highly toxic to mononuclear cells, and that it down-regulates the expression of the key phagocytic marker MERTK at the protein level (preliminary data, study is ongoing). In summary, the data presented in this thesis suggests that the CD40 risk gene and the immunosuppressant Cladribine play roles in altering the phenotype in mononuclear cells. The understanding from this body of work provides some insights into how genetic susceptibility can drive changes in mononuclear cell phenotype which may impact on the modulation of co-stimulatory mechanisms in humoral immunity. Moreover, the findings from the Cladribine study may assist the field in the discovery of alternative mechanisms of the drug on mononuclear cells and therefore inform a refined approach to treatment of MS.
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ItemSpatial and temporal surveillance of the mechanisms controlling proteome foldedness via a FRET-based biosensor( 2018)Proteostasis (protein homeostasis) is essential for keeping the proteome functional. This process controls protein synthesis, folding and degradation and involves hundreds of genes, including those encoding chaperones, to form extensive quality control (QC) networks (Kim et al., 2013). Imbalances in proteostasis are implicated in a range of aggregation-based neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Huntington’s and Alzheimer’s diseases (Morimoto et al., 2014; Vilchez et al., 2014). Currently there is a lack of capacity to quantitatively measure proteostasis imbalance and therefore we are limited in understanding how proteostasis imbalance manifests during disease. A new biosensor system has been developed by our lab to address this shortfall. The biosensor is a genetically encoded unfolded “bait” flanked by two fluorescent proteins to assay foldedness by fluorescence resonance energy transfer (FRET). Proteostasis efficiency is reported by measurement of the efficiency to which the bait interacts with the QC network. In this master’s project, the biosensor was further targeted to organelles to allow for a higher degree of spatiotemporal control. Signalling peptides were used to target the biosensor to specialised microenvironments, and successful targeting was achieved in the Golgi apparatus and nucleus. Investigations into nuclear proteostasis revealed the biosensor behaved predictably to chaperone overexpression (Hsp40 and Hsp70 co-expression) or inhibition (Hsp70 or Hsp90 inhibition). Polyglutamine (PolyQ) expansions of non-pathogenic (Q25) to pathogenic (Q72) lengths reduced the biosensor foldedness and decreased aggregation, which is consistent with an increase in chaperone supply. The biosensor was also adapted to express in the body wall muscles of Caenorhabditis elegans to examine change in proteostasis across age and in an organismal context. The biosensor was successfully expressed in the model organism, with potential sub-microscopic and variant biosensor expression level confounding data analysis. The C. elegans reporter lines were successfully crossed with lines expressing Aβ (1-42) demonstrating the ability of the biosensor to report on disease states. Moving forward, the generation of low-expression, single-copy C. elegans biosensor lines would allow for steady, matched expression and enhanced capacity for comparison between worm lines.
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ItemTryptophan hydroxylase-2 immunoreactivity changes in the dorsal raphe nucleus after chronic methamphetamine or corticosterone in BDNF-deficient mice models: Implications for schizophrenia( 2019)Schizophrenia is a major mental illness caused by genetic and environmental factors. In addition to dopaminergic dysfunction, evidence has associated 5-hydroxytryptamine (serotonin) with schizophrenia. The serotonergic system interacts with the neurotrophin, Brain-Derived Neurotrophic Factor (BDNF), resulting in behavioural and anatomical changes relevant to schizophrenia. The aim of this study was to determine changes in the number of serotonergic cells within the dorsal raphe nucleus (DR) in BDNF-deficient mice (BDNF heterozygous and BDNF Val66Met polymorphism) after chronic treatment with methamphetamine (METH) or the stress hormone, corticosterone (CORT). Chronic METH abuse may lead to psychosis and dopaminergic dysfunction. Chronic stress is a risk factor for schizophrenia in vulnerable individuals. Cell quantification and density analysis were based on the immunoreactivity of the serotonin-synthesizing enzyme, tryptophan-hydroxylase 2 (TPH2), within subnuclei of the DR: DR caudal (DRC), DR dorsal (DRD), DR interfascicular (DRI), DR ventral (DRV) and DR ventrolateral (DRVL) subnuclei. Results can be summarized as follows: 1) METH was able to decrease the number of TPH2-immunoreactive (TPH2-ir) cells in both BDNF-deficient models within the medial and caudal DRVL subnuclei; 2) BDNF-deficiency resulted in reductions in the number of TPH2-ir cells in a subnucleus- and treatment-specific way; namely BDNF heterozygosity induced a reduction in METH-treated mice within the DRVL subnucleus, whilst the BDNF Val66Met polymorphism resulted a reduction of TPH2-ir neurons in vehicle-treated mice within the caudal part of the DRVL subnuclei. The latter finding was also observed through density analysis; 3) CORT treatment increased TPH-ir neurons in the rostral DRV and the caudal DRVL of Val/Met mice; and 4) Noticeably, there were no changes within the DRD, DRC or DRI subnuclei regardless of the BDNF-deficiency or treatment. These results suggest that the number of TPH2-ir cells is dependent on BDNF levels as well as on a history of stress or methamphetamine abuse. These changes induced by BDNF-deficiency or the treatments are subnucleus-specific, affecting more the rostral, caudal and lateral edges rather than core and dorsal areas of the DR. The results suggest that these subnuclei-specific effects may have implications for brain regions dif-ferentially innervated by these subnuclei, including the amygdala, hippocampus and periaqueductal gray, which are involved in schizophrenia and mood disorders.
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ItemApplying matrix assisted laser desorption-ionization mass spectrometry to identify and quantify isoforms of amyloid beta in human brain( 2017)Amyloid beta (Aβ) plays a central pathogenic role in Alzheimer's Disease (AD), however its great heterogeneity remains largely unexplored as most research focuses on only two specific species: Aβ1-40 and Aβ1-42. More than fifty truncated species of amyloid beta have been identified, as well as a multitude of additional post translational modifications including pyroglutamation, oxidation and glycosylation. This spectrum of isoforms is either cumbersome or impossible to resolve with prevailing methods such as ELISA and western blots. Matrix Assisted Laser Desorption-Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) allows specific and simultaneous identification of these species. My thesis employed this method, with prior enrichment of donated brain samples with amyloid beta immunoprecipitation, to characterise these isoforms in different fractions of human brain: the cytosolic (TBS) fraction, the peripheral membrane/vesicular (Na2CO3) fraction, the integral lipid/membrane (urea/detergent) fraction, and the polymerised fibrillary (formic acid) fraction. At least fourteen different Aβ isoforms were resolved, most in the formic acid fraction and membrane fraction, with relatively less in the sodium carbonate fraction. Extending the methodology, isotopically labelled ‘heavy’ internal standards were utilised to allow for absolute quantification of some prominent truncations, including Aβ4-42 and Aβ1-42. This showed a mean of 704 pmol/g wet brain of Aβ4-42 in formic acid fractions of brains with Alzheimer’s pathology, and 438 pmol/g in the membrane fractions of brains with Alzheimer’s pathology. Aβ1-42 was present in the formic acid fractions and membrane fractions of those with Alzheimer’s Disease in mean values of 1.3 nmol/g and 232 pmol/g respectively. These values were greater than those in the control brains. It was also seen that Aβ4-42 ionised approximately 1.3 fold the extent of AβH1-42, and approximately 3 fold that of AβH1-40. Noting the difficulties inherent in ionising entire species of amyloid beta, enzymatic cleavage with Lys-N was undertaken to improve reliability and throughput of MALDI-TOF MS analysis. Lys-N is a metalloendopeptidase that preferentially cleaves N-terminals of lysine groups. These cleaved products of Aβ are more amenable to MALDI-TOF MS than products of C-terminal cleavage. Lys-N was first isolated from the fruiting bodies of pleurotus ostreatus, achieving a 3.94 fold purification. This was then applied to synthetic amyloid beta and MALDI-TOF MS analysis, preferentially resolving N terminal truncated isoforms of Aβ. With further refinement, this method would allow a specific and robust approach to characterising and quantifying multiple species of Aβ to picomole amounts.
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ItemCharacterization and stress induced modulations of RXFP3+ neurons in the mouse BNST( 2017)Stress induced relapse and stressful emotional responses occurring during drug withdrawal are common problems for drug addicts. The relaxin-3/relaxin family peptide receptor 3 (RXFP3) system modulates stress responses and reward seeking behaviour. RXFP3 is expressed within the bed nucleus of the stria terminalis (BNST), and bilateral intra-BNST injections of an RXFP3 antagonist decreased self-administration and stress induced reinstatement of alcohol-seeking in rats. The electrophysiological properties of RXFP3+ neurons in the BNST and whether they are responsive to stress are presently not known. Similarly, the possibility of convergent signalling with other neuropeptides such as corticotropin releasing factor (CRF) in the BNST is not clear either. Therefore, whole cell recordings were conducted on defined RXFP3+ neurons in brain slices taken from adult RXFP3-Cre x tdTomato mice (n=38 mice for naive group). To study any alterations in RXFP3+ dorsal BNST neurons induced by chronic stress, a group of mice (n=13) were subjected to swim stress daily for 5 days. Mice were anaesthetised (5% isoflurane), decapitated and 250 µm coronal slices through the BNST were obtained. Based on Hammack’s characterization, the majority of recorded RXFP3+ neurons (~75%) were classified as Type II neurons and a new type of neuron, Type IV neurons were found within the dorsal BNST. Surprisingly, the electrophysiological profile of RXFP3+ neurons was not fixed; five days of swim stress induced plasticity, shifting a population of Type II neurons to Type III or Type IV neurons. Notably, stress induced plasticity of BNST RXFP3+ neurons was not only found in terms of neuron types, but also in their responsiveness to afferent inputs. The decay time of both spontaneous inhibitory postsynaptic currents (P=0.0005, Mann-Whitney test) and spontaneous excitatory postsynaptic currents (P=0.0028, Mann-Whitney test) were significantly increased after stress (naive, n=70 cells from 38 mice; stress, n=32 cells from 13 mice). Most BNST RXFP3+ neurons from the naive group (6 out of 9) were hyperpolarised following bath application of a selective RXFP3 agonist (A2, 100 nM), in line with an inhibitory role of this neuropeptide. Bath application of CRF (100 or 300 nM) onto brain slices during gap free voltage clamp recordings were conducted (n=14) to examine potential interactions between these peptide systems. CRF induced an increase of GABA release onto RXFP3+ neurons (~40% of the RXFP3+ neurons recorded), but decreased GABA release for ~35% of RXFP3+ neurons. In addition, CRF also caused direct postsynaptic effects on RXFP3+ neurons. Overall, these experiments have helped to elucidate the electrophysiological properties of RXFP3+ BNST cells, their responsiveness to stress, their interaction with a prototypic stress neuropeptide and their potential role in stress induced reward-seeking.
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ItemMinimally invasive deep brain stimulation via an endovascular stent-electrode array( 2016)Modern neural interfaces are practical demonstrations of what multi-disciplinary teams can create in this age of medicine. From decoding brain signals in order to control prosthetic limbs, to stimulating peripheral nerves in amputees to restore sensation, the blend of engineering and medicine continues to generate novel, life-changing, therapeutics. In an attempt to create a paradigm shift towards minimally invasive implantation of such neural interfaces, the Vascular Bionics Laboratory at The Royal Melbourne Hospital has designed a stent-electrode array that can be deployed in the cerebrovasculature via a series of percutaneously positioned catheters. While the device was designed to record brain signals for the purpose of decoding them, its stimulation capabilities had not been studied. This thesis takes that next step by conducting stimulation studies with the device. Were such a stimulation process established with these proof of concept experiments, then the device would hold promise for future neural interfaces. The animal model that the stent-electrode arrays had been designed for was used for these stimulation studies. Seven Corriedale sheep had devices implanted, and then a series of stimulation experiments were conducted across a range of time points using a variety of stimulation parameters and configurations while the animals were awake; the objective of stimulation was to excite the motor cortex and thereby induce a categorical response, such as the contraction of a limb. Stimulation parameters included monophasic, either anodal or cathodal, pulses at phase widths of 200 µs or 500 µs and at frequencies of either 20 Hz or 50 Hz with currents ranging from 1 to 6 mA. These were applied across a variety of configurations including monopolar, with either a head-mounted or flank-mounted return electrode, and bipolar. Pulse trains were also studied. Induced motor activity was documented principally by visual observation and accelerometer tracking. A range of motor activity was documented across the animals including about the flank, the neck, the ear, and some facial muscles. However, these contractions did not align to the neuroanatomical position of the electrodes relative to the motor cortex, nor were the contractions consistent across time points. In addition, the majority of contractions were not elicited from bipolar stimulation and thus prone to induction by current spread. Four devices were found on autopsy to have severed cables connecting to the stent-electrode arrays, a failure not identified by the regular tacking of impedances in the device. The dataset generated from these stimulation studies suggested that the motor activity documented was a product of current spread through the devices to muscles of the sheep, rather than motor activity caused by stimulation of the motor cortex. These studies serve to guide future experiments with this device.